Practical 08 Introduction to taxonomic analysis

Introduction to taxonomic analysis

of amplicon and shotgun data using
QIIME
September 2014
Author Peter Sterk
Oxford e-Research Centre, University of Oxford, UK/
European Bioinformatics Institute, Hinxton, UK
Contributors: BPA-CSIRO Metagenomics trainers

Table of Contents
Introduction to taxonomic analysis of amplicon and shotgun data using QIIME ..... 1
General information .......................................................................................... 3
Resources used .................................................................................................. 3
Tutorial objectives ............................................................................................................................................. 4
Short introduction to Linux ........................................................................................................................... 4
De novo OTU picking and diversity analysis using 454 data .................................... 7
Prepare files .......................................................................................................................................................... 7
To denoise or not to denoise? ..................................................................................................................... 10
Picking Operational Taxonomic Units (OTUs) ................................................................................... 10
View OTU statistics ......................................................................................................................................... 11
Visualize taxonomic composition ............................................................................................................. 11
Alpha diversity within samples and rarefaction curves ................................................................. 12
Beta diversity and beta diversity plots ................................................................................................... 13
Closed reference OTU picking of 16S ribosomal rRNA fragments selected from a
shotgun data set .................................................................................................. 13
Extraction of 16S rRNA sequence-containing reads with rRNASelector ................................ 13
Closed-reference OTU picking workflow and visualization of results in Megan 5 .............. 15
Extreme challenge ................................................................................................ 16
Finally .................................................................................................................. 16

General information
The following standard icons are used in the hands-on exercises to help you
locating:

Important Information
General information / notes
Follow the following steps
Questions to be answered
Warning PLEASE take care and read carefully
Optional Bonus exercise

Resources used
QIIME (http://qiime.org/index.html)
Sutton et al. (2013). Impact of Long-Term Diesel Contamination on Soil Microbial
Community Structure. Appl. Environ. Microbiol. 79(2):619-630.
Sutcliffe et al. (2013). Draft Genome Sequence of Thermotoga maritima A7A
Reconstructed from Metagenomic Sequencing Analysis of a Hydrocarbon Reservoir
in the Bass Strait, Australia. Genome Announc. 1(5): e00688-13.
Li et al. (2013). Draft Genome Sequence of Thermoanaerobacter sp. Strain A7A,
Reconstructed from a Metagenome Obtained from a High-Temperature Hydrocarbon
Reservoir in the Bass Strait, Australia. Genome Announc. 1(5): e00701-13.
Lee et al. (2011). rRNASelector: a computer program for selecting ribosomal RNA
encoding sequences from metagenomic and metatranscriptomic shotgun libraries. J.
Microbiol. 49(4):689-691.

Tutorial objectives
In this tutorial we will look at the open source software package QIIME (pronounced
chime). QIIME stands for Quantitative Insights Into Microbial Ecology. The
package contains many tools that enable users to analyse and compare microbial
communities. QIIME was originally developed to analyse of Roche 454 amplicon
sequencing data. In the latest versions workflows have been added to analyze data
from different sequencing platforms, such as Illumina, and different types of data,
such as shotgun data. In this course we will use QIIME 1.8, which is the latest version.
We will (re-)introduce you to the Linux operating system to a basic level that is
sufficient to run bioinformatics software from preconfigured Linux installations such
as the one we will be using today.
After completion of this tutorial, you should be able to perform a taxonomic analysis
on a Roche 454 16S rRNA amplicon dataset. In addition you should be able to do 16S
taxonomic analysis on shotgun data using the tool rRNASelector in combination with
QIIME.
Finally you should be able to work out solutions for datasets from other platforms
such as Illumina from the information you find on the QIIME web site
(http://qiime.org/).

Short introduction to Linux

QIIME runs under Linux or Mac OS X, but not under Windows. This tutorial was
written for the QIIME installation installed on the Ubuntu Linux virtual machine
image that was created for this course.
It is worth mentioning at this stage that if you find QIIME useful for your own project,
but you lack the expertise to set up your own Linux system, the QIIME developers
produce a downloadable disk image that contains the full QIIME package. You will
need to install VirtualBox, which is freely available for Linux, Mac and Windows
platforms and this will allow you to run QIIME on any of these platforms as a virtual
machine. More details can be found at http://qiime.org/install/virtual_box.html.
Another alternative is the Bio-Linux image (http://nebc.nerc.ac.uk/tools/bioLinux/bio-Linux-7-info). It is a Linux distribution containing many bioinformatics
packages, runs live from DVD or memory stick, but can also be installed on an
computer, either as the main operating system or under VirtualBox or similar (e.g.
VMWare, Parallels). It has the basic packages from QIIME installed, but depending
on your requirements you may need to install additional components. It comes with a
very good Linux tutorial, so worth investigating if you want to go that route. Finally,
you can run QIIME in the Amazon EC2 cloud. For more details, see
http://qiime.org/tutorials/working_with_aws.html#working-with-ec2.
As a number of you will not have any prior experience with Linux, this part of the
tutorial will teach you the basics of Linux. There are a large number of free Linux
tutorials on the web for those who want to learn more at a later stage.

We assume you have successfully booted your computer into Linux and you have the
Linux desktop on your screen.
The first steps well do together now to get you going as quickly as possible. One
piece of advice, try to type the commands into your terminal rather than copy and
paste as it will help you understand the commands better. Also, Microsoft Word has
replaced certain characters, e.g. straight quotes with smart quotes; these and a number
of other characters cause trouble.
Most of what we will do will be run from the command line and before we can issue
any commands, we will need a terminal window. Click on Applications at the top left
of your desktop, then go to Accessories and then click on the first Terminal menu
item. A terminal window should appear on your desktop.
At the prompt (which ends with $) you can type commands. During this tutorial we
will represent the prompt as $ for brevity, do not type a dollar character at the
beginning of any of the commands, only type what follows it. To execute a command,
press return/enter.
Type the following command to list the files and directories (folders) followed by
enter:
$ ls

You will be presented with a list of the contents of your home directory. Note that
Linux does not have a concept of disks like windows (e.g. C:\). Instead it has so called
mount points with a directory at its root. /home is where by default the home
directories of all users are located. /usr is where a lot of the operating system and
programs reside. The directory structure of a Linux system outside the /home area is
for this tutorial not important. Also note that where windows uses back slashes to
separate directories, Linux uses forward slashes. The desktop is in a folder
/home/trainee/Desktop.
To move up to the desktop folder, type
$ cd Desktop
$ ls -l

Note that file and directory names are case-sensitive, cd desktop does not work. The
-l option after the ls command tells ls to show a long, more detailed listing of the
directory contents showing file permissions, owners and date stamps. On your
desktop is a folder called Taxonomy, which contains the necessary files for this
tutorial. You can probably work out how to enter this directory now. There are a few
more tips for moving around:
To go to your home directory, type one of the following (note ~ is short for your
home directory):
$ cd
$ cd ~

To go to the tutorial directory, type one of the following:

$ cd /home/trainee/Desktop/Taxonomy
$ cd ~/Desktop/Taxonomy
$ t

The command t is an alias that we have set up to make life easier. You can create
your own aliases for command that you use frequently using alias, e.g.
$ alias d=cd ~/Desktop

From that moment on, you only need to type d followed by Enter to go to your
desktop folder.
To move up one directory level (e.g. when you are in Taxonomy and want to go back
to Desktop), type:
$ cd ..

.. is the parent directory, . Is the current directory.

If youre not sure where you are on the file system, type pwd to print the current
working directory to the terminal window.
A few more useful commands:
To view text files (or concatenate multiple files into a new file):
$ cat filename1 (filename2 filename 3 . > newfile

To view long text files one screen at a time, use less. Exit less by typing q at the
colon.
$ less filename

# less S will truncate long lines

To copy a file:
$ cp file newfile

# results in two identical files called file and newfile

To move or rename a file:

$ mv file newfile

# renames the file to newfile

$ mv file ..

# moves the file to the parent directory

To remove/delete a file:
$ rm file

# be cautious, in Linux a deleted file is gone forever

1
2

For now this is all we need to know to start the tutorial proper.

De novo OTU picking and diversity analysis using 454

data
We will partly re-analyze the data from Sutton et al. (2013). Impact of Long-Term
Diesel Contamination on Soil Microbial Community Structure. Appl. Environ.
Microbiol. 79(2):619-630. An electronic copy of the paper can be found in your
Taxonomy folder. When you have to wait a few minutes for commands to complete,
use the time to acquaint yourself with the study. It is a good example of a study that
combines the power of next-generation sequencing with environmental
observations/measurements.
The analysis we do follows the pipeline described in the QIIME general 454 tutorial
(http://qiime.org/tutorials/tutorial.html). Feel free to look at this tutorial for further
background information. As our dataset used in the tutorial is a subset of the Sutton
data, but more realistic for what you may be doing. In some parts of the analysis and
steps we have precomputed analysis on the complete data set for comparison.
Go to /home/trainee/Desktop/Taxonomy/sutton/. You will find a file called
sutton5000.sff, which contains the 5000 random multiplexed reads from this studys
26 samples with quality information and flowgrams. This is a binary file and we will
need to extract the sequences and quality scores from it to be able to work with the
sequences and quality scores. We will use sffinfo from Roche to do this. This tool is
not supplied with QIIME, but it is freely available from Roche on request. We have
installed it for you. Alternatively, you can use the much slower tool with the same
functionality supplied with QIIME.
Note that in the Taxonomy folder there is a subfolder called sutton_full_denoised.
This folder contains the precomputed analysis results from the full and denoised
dataset. When you go through this tutorial, it is worth comparing those results with
the results you obtain with the reduced dataset.
Prepare files
For this tutorial we have generated a random subset of the original Sutton dataset to
complete the necessary steps in a reasonable amount of time.
Generate a fasta and quality score file from sutton.sff:
$ sffinfo -s sutton5000.sff > sutton.fna
$ sffinfo -q sutton5000.sff > sutton.qual

Note that the > character redirects the output from the screen to a new file. Please
inspect the files with less or less -S to truncate long lines. You will notice that
there is no obvious association of the reads with a particular sample.

That is what we need to do next.

$ less -S sutton.fna

Or just have a look at the fasta headers and ignore the DNA sequences with the
command grep. You need to extract all lines that start with > and send the output
to less to be able to view the output one screen at a time. The command to give is:
$ grep ^> sutton.fna | less

^> is a so-called regular expression. The ^ character means starts with, so the
grep command looks for all lines that start with >. The pipe character, | is used to
pipe or stream the output from the first command (grep) into a second command (less).
Now view the file containing the quality scores:
$ less -S sutton.qual

The Mapping File.

We will use the barcode information to associate reads with one of 26 samples. We
will also need to remove the barcodes and primer sequences from the reads as these
interfere with the taxonomic analysis.
You will find a file called mapping.txt in the Taxonomy/sutton/ directory.
Note that this file has to be created for each analysis, as the information is specific for
an experiment. The mapping file can also contain information on your experimental
design. The format is very strict; columns are separated with a single TAB character;
the header names have to be typed exactly as specified in the documentation. A good
sample description is useful as it is used in the legends of the figures QIIME generates.
We could also specify the reverse primer and remove it from the reads. Unfortunately,
the reverse primer sequence was not in the paper, and well ignore it though we could
probably deduce it from longer reads: as a 466 bp region of the 16S ribosomal RNA
gene flanking the V3 and V4 regions was amplified, youll have a clue where to look
for the reverse primer.
$ less -S mapping.txt
#SampleID
BarcodeSequence LinkerPrimerSequence
Description
A1
ACATACGCGT
CCTAYGGGRBGCASCAG
A1_Fill_Polluted
A2
ACGCGAGTAT
CCTAYGGGRBGCASCAG
A2_Fill_Polluted
B1
ACTACTATGT
CCTAYGGGRBGCASCAG
B1_Fill_Clean
B2
ACTGTACAGT
CCTAYGGGRBGCASCAG
B2_Clay_Polluted
B3
AGACTATACT
CCTAYGGGRBGCASCAG
B3_Peat_Polluted
B4
AGCGTCGTCT
CCTAYGGGRBGCASCAG
B4_Peat_Polluted
C1
AGTACGCTAT
CCTAYGGGRBGCASCAG
C1_Fill_Clean
C2
ATAGAGTACT
CCTAYGGGRBGCASCAG
C2_Peat_Clean
C3
CACGCTACGT
CCTAYGGGRBGCASCAG
C3_Peat_Polluted
D1
CAGTAGACGT
CCTAYGGGRBGCASCAG
D1_Fill_Clean
D2
CGACGTGACT
CCTAYGGGRBGCASCAG
D2_Clay_Clean
D3
TACACACACT
CCTAYGGGRBGCASCAG
D3_Clay_Polluted
D4
TACACGTGAT
CCTAYGGGRBGCASCAG
D4_Peat_Polluted

D5
E1
E2
F1
F2
G1
G2
G3
H1
H2
H3
I1
I2

TACAGATCGT
TACGCTGTCT
TAGTGTAGAT
ACAGTATATA
ACGCGATCGA
TCTAGCGACT
TCTATACTAT
TGACGTATGT
TACTCTCGTG
TAGAGACGAG
TCGTCGCTCG
TCGATCACGT
TCGCACTAGT

CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG
CCTAYGGGRBGCASCAG

D5_Sand_Polluted
E1_Fill_Clean
E2_Fill_Polluted
F1_Sand_Clean
F2_Sand_Polluted
G1_Fill_Clean
G2_Fill_Clean
G3_Fill_clean
H1_Peat_Clean
H2_Peat_Clean
H3_Sand_Clean
I1_Sand_Clean
I2_Sand_Clean

Next test the mapping file for potential errors:

$ validate_mapping_file.py -m mapping.txt -o mapping_output

There shouldnt be any errors. If there are errors, a corrected mapping file will be
written to the directory mapping_output.
Assign samples to the reads
Using the mapping file and the Sutton fasta and quality files we are going to now
assign samples to the reads.
Type the following command on a single line:
$ split_libraries.py -m mapping.txt f sutton.fna q sutton.qual o
split_library_output b 10 L 500

Here we specify the following options:

-m for the mapping file
-f for the fasta input file generated from the .sff file
-q for the quality score file generated from the .sff file
-o for the output directory
-b for the length of the barcode (10 in our case)
-L for the maximum length of the read we still accept. Useful if you do not know the
reverse primer, but you do know the approximate length of the sequence you have
amplified. Reads that are much longer are probably an artifact and it is best to exclude
them.
When completed please enter the directory split_library_output and have a look at
the log file. It contains detailed information what was done during the step weve just
performed. Note that the number of reads assigned to the different samples varies
considerably. Knowing where the individual samples were taken may give a clue why
this may be! Next have a quick look at the file seqs.fna. What has changed to the
header of the reads?
Look at the split libraries results
$ cd split_library_output
$ ls -l
$ less split_library_log.txt

$ less -S seqs.fna

You will see the reads are now batched to their sample.
To denoise or not to denoise?
The pyrosequencing technology employed by 454 sequencing machines produces
characteristic sequencing errors, mostly imprecise signals for longer homopolymers
runs. Most of the sequences contain none or only a few errors, but a few sequences
contain enough errors to be classified as an additional rare OTU. The goal for the
denoising procedure is to reduce the number of erroneous OTUs and thus increasing
the accuracy of the whole QIIME pipeline. This is a computationally intensive
procedure, which we will skip for this reason. There is a QIIME tutorial that outlines
the steps (http://qiime.org/tutorials/denoising_454_data.html) and also includes a
warning about new 454 flow patterns introduced in 2012. Note that only amplicon
data sets can be denoised with the described procedure. We will not denoise our data
today. We did denoise the full dataset in the sutton_full_denoised folder.
Picking Operational Taxonomic Units (OTUs)
We will now use a workflow for de novo OTU picking, taxonomy assignment,
phylogenetic tree construction, and OTU table construction QIIME has several
workflows to pick OTUs, we will be using the one described in the general overview
tutorial (http://qiime.org/tutorials/tutorial.html) It has 7 steps, which are described in
some detail in this tutorial.
The described procedure is run with the command from the Taxonomy directory. This
step takes about 12mins to run. Please read through the different steps
(http://qiime.org/tutorials/tutorial.html) and try to understand the procedure.
Remember that an OTU is not the same as a species, but a bag/cluster of highly
similar sequences (at least 97% is common for bacteria/archaea), or a single sequence
in case of rare OTUs.
$ pick_de_novo_otus.py -i split_library_output/seqs.fna -o otus

Please do spend some time looking at the output of this pipeline. In particular the file
seqs_rep_set_tax_assignments.txt in the uclust_assigned_taxonomy directory. By
default QIIME uses the Greengenes 16S reference database to assign taxonomy. It has
the following levels: kingdom, phylum, class, order, family, genus, species. It will be
immediately clear that most reads cannot be classified up to species level.
As described in step 6 of the QIIME overview tutorial, the pipeline creates a Newickformatted phylogenetic tree (rep_set.tre) in the otus directory. You can run the
program figtree either from the command line or select FigTree from the menu on
your desktop (Applications -> Other -> FigTree) and view the tree by opening the file
rep_set.tre in the otus folder (Desktop->Taxonomy->otus). The tree that is
produced is too complex to be of much use. We will look at a different tool, Megan 5,
which produces a far more useful tree.
10

In step 7 of the QIIME overview tutorial a file called otu_table.biom is generated. It is

in biom-format, which is increasingly supported by taxonomic software developers.
One of the tools that supports the biom format is Megan (http://ab.inf.unituebingen.de/software/megan5/). Megan is a standalone tool for analyzing both
taxonomic and functional content of datasets. It is free for academic use, but you will
need to request a licence first. We will use Megan version 5 to display a taxonomic
tree using the biom output we have just produced.
Note: Sequence errors can give rise to spurious ORFs and we can filter out OTUs that
only contain a single sequence (singletons). QIIME allows you to do this quite easily
or you could also remove abundant taxa if you are more interested in rare taxa. To
remove singletons, run the following commands:
$ cd otus
$ filter_otus_from_otu_table.py -i otu_table.biom
-o otu_table_no_singletons.biom -n 2

This removes OTUs with less than 2 sequences. If you use the k option instead of the
n option, OTUs with more than the specified number of sequences will be removed.
Megan can be opened from the menu under Applications -> Other. From the File
menu select Import -> BIOM format. Find your biom file and import it. Megan will
generate a tree that is far more informative than the one produced with FigTree. You
can change the way Megan displays the data by clicking on the various icons and
menu items. Please spend some time exploring your data. The Word Cloud
visualization is interesting, too, if you want to find out which samples are similar and
which samples stand out.
View OTU statistics
You can generate some statistics, e.g. the number of reads assigned, distribution
among samples. Some of the statistics are useful for further downstream analysis, e.g.
beta-diversity analysis. Run the following now, again from within the Taxonomy
directory, and look at the results. Write down the minimum value under
Counts/sample summary. We need it for beta-diversity analysis.
$ cd ../
$ biom summarize-table -i otus/otu_table.biom o
otus/otu_table_summary.txt
$ less otus/otu_table_summary.txt

Visualize taxonomic composition

We will now group sequences by taxonomic assignment at various levels. The
following command produces a number of charts that can be viewed in a browser.
The command takes about 5 minutes to complete.

11

$ summarize_taxa_through_plots.py -i otus/otu_table.biom -o
wf_taxa_summary -m mapping.txt

To view the output, open a web browser from the Applications -> Internet menu. You
can use Google chrome, Firefox or Chromium.
In Google chrome or Chromium, type CTRL-O, or in Firefox use the File menu to
select Desktop ->Taxonomy -> wf_taxa_summary -> taxa_summary_plots and open
either area_charts.html or bar_chars.html. I prefer the bar charts myself. The top chart
visualizes taxonomic composition at phylum level for each of the samples. The next
chart goes down to class level and following charts go another level up again. The
charts (particularly the ones more at the top) are very useful for discovering how the
communities in your samples differ from each other. There is a similar plot in the
paper, if you have time, see how our analysis compares with the one described in the
paper.
Alpha diversity within samples and rarefaction curves
Alpha diversity is the microbial diversity within a sample. QIIME can calculate a lot
of metrics, but for our tutorial, we generate 3 metrics from the alpha rarefaction
workflow: chao1 (estimates species richness); observed species metric (the count of
unique OTUs); phylogenetic distance. The following workflow generates rarefaction
plots to visualize alpha diversity.
Run the following command from within your taxonomy directory, this should take a
few minutes:
$ alpha_rarefaction.py -i otus/otu_table.biom -m mapping.txt -o
wf_arare -t otus/rep_set.tre

First we are going to view the rarefaction curves in a web browser by opening
/home/trainee/Desktop/Taxonomy/wf_arare/alpha_rarefaction_plots/rarefaction_plots.
html.
To start select as metric chao1 and select as category Description. It is clear that
the microbial diversity in some samples is much higher than in other samples. Click
around in the legend as this will help you work out which line corresponds with which
sample. If you have time you could try to correlate species richness with
environmental data from the paper and establish whether our analysis confirms the
findings of the authors.
Next view the precomputed rarefaction curves which show an increased sequencing
depth.
In general the more reads you have, the more OTUs you will observe. If a rarefaction
curve start to flatten, it means that you have probably sequenced at sufficient depth, in
other words, producing more reads will not significantly add more OTUs. If on the
other hand hasnt flattened, you have not sampled enough to capture enough of the
microbial diversity and by extrapolating the curve you may be able to estimate how
many more reads you will need. Consult the QIIME overview tutorial for further
information.

12

Beta diversity and beta diversity plots

Before we have a quick look at taxonomic analysis of shotgun data, we have a quick
look at beta diversity analysis, which is the assessment of differences between
microbial communities. As we have already observed, our samples contain different
numbers of sequences.
The first step is to remove sample heterogeneity by randomly selecting the same
number of reads from every sample. This number corresponds to the minimum
number recorded when you looked at the OTU statistics.
Now run the following command
$ beta_diversity_through_plots.py -i otus/otu_table.biom -m
mapping.txt -o wf_bdiv_even122 -t otus/rep_set.tre -e 122

Read through the beta diversity compute section of the QIIME overview tutorial and
try to understand this workflow. We will look at visualization of beta diversity
analysis results in more detail in the next tutorial focusing on visualization.

Closed reference OTU picking of 16S ribosomal rRNA

fragments selected from a shotgun data set
In a closed-reference OTU picking process, reads are clustered against a reference
sequence collection and any reads, which do not hit a sequence in the reference
sequence collection are excluded from downstream analyses. In QIIME,
pick_closed_reference_otus.py is the primary interface for closed-reference OTU
picking in QIIME. If the user provides taxonomic assignments for sequences in the
reference database, those are assigned to OTUs. We could use this approach to
perform taxonomic analysis on shotgun data. We need to perform the following steps:
1. Extract those reads from the data set that contain 16S ribosomal RNA
sequence. If there are less than (e.g.) 100 nucleotides of rRNA sequence, the
read should be discarded.
2. Remove non-rRNA sequence (flanking regions) from those reads
3. Run closed-reference OUT picking workflow
4. Visualise the results, e.g. in Megan
Extraction of 16S rRNA sequence-containing reads with rRNASelector
We will analyze an Illumina paired-end dataset that has been drastically reduced in
size for this tutorial, while preserving the majority of the 16S containing reads. The
dataset
is
from
the
metagenome
described
at
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772140/. There is a pdf in the
working directory for this part of the tutorial. This is a paired end dataset, and where
read pairs overlapped, they were merged into a single sequence. If read pairs did not
overlap, both reads were included in the analysis. QC was performed using the EBI
Metagenomics pipeline.

13

We will use a tool called rRNASelector, which is freely available

(http://www.ncbi.nlm.nih.gov/pubmed/21887657) to select our 16S rRNA sequence
containing reads. The tool invokes hmmsearch and uses trained hidden Markov
models to detect reads with 16S rRNA sequence. The tool also trims the reads so that
only 16S rRNA sequence is present in the fasta file we will feed into the QIIME
workflow.
First, we need to go to our working directory:
% cd ~/Desktop/Taxonomy/A7A/

You will find a file called A7A-paired.fasta containing the sequence reads.
Fire up rRNASelector from the command line:
% rRNASelector

A graphical interface should appear. Load the sequence file by clicking on File
Choose at the top and navigate to the file A7A-paired.fasta. Select the file and click
Open. The tool will automatically fill in file names for the result files. Change the
Number of CPUs to 2, select Prokaryote 16S (to include both bacterial and archaeal
16S sequences) and specify the location of the hmmsearch file by clicking the second
File Choose button. You can type the location manually /usr/bin/hmmsearch. Next,
click process. The run should take a few minutes to complete.

14

If all went well, you can close rRNASelector by clicking on Exit. You will have 3
new files in your directory, one containing untrimmed 16S reads, one containing
trimmed 16S reads (A7A-paired.prok.16s.trim.fasta; thats the one we want) and a file
containing reads that do not contain (sufficient) 16S sequence.
Closed-reference OTU picking workflow and visualization of results in
Megan 5
We are now ready to pick our OTUs. We do that by running the following command
(all on one line and no space after gg_otus-12-10):
% pick_closed_reference_otus.py -i A7A-paired.prok.16s.trim.fasta
-o ./cr_uc
-r /mnt/workshop/tools/qiime_software/gg_otus-12_10release/rep_set/97_otus.fasta
-t /mnt/workshop/tools/qiime_software/gg_otus-12_10release/taxonomy/97_otu_taxonomy.txt

15

We need to specify the following options:

-i input_file.fasta
-o output_directory
-r /path/to/reference_sequences
-t /path/to/reference_taxonomy
The command will take several minutes to run. When finished open Megan as
described before, import the otu_table.biom file and explore the results.

Extreme challenge
Many of you will send off samples to be sequenced and quite often providers
preprocess the raw data before handing it back to the client. If your reads were
demultiplexed and primers were removed, you have a problem as the amplicon
workflows from QIIME rely on the presence of primers and barcodes. The first
message is that you should insist on getting your data as unprocessed reads. Not all is
lost if you end up with a dataset with primers and barcodes stripped, but it requires
more work.
This is an exercise in understanding the format QIIME expects and how you can
reformat data to allow analysis in QIIME.
There is a directory in your Taxonomy folder called Baltic, with data from the
Baltic Sea. It has a number of samples that were sequenced using 454 technology and
are demultiplexed, but for the purpose of this exercise, these could have been Illumina
files as the format is fastq.
The challenge we give you is to write down how we need to reformat this data (even
if you do not know how) to be able to perform a similar analysis we have done with
the Red Sea data. This is something you can in a small group if you feel youre not
quite up to the challenge.
Hints:
Consider using the QIIME function convert_fastaqual_fastq.py
What does the mapping you need to run split_libraries.py look like?

Finally
If there is time left you could go back to the polluted railway site study. The aim of
this study was to understand interrelationship among microbial community
composition, pollution level, and soil geochemical and physical properties. With
additional information from the paper, could you come up with some conclusions?
The QIIME 454 overview tutorial at http://qiime.org/tutorials/tutorial.html has a
number of additional steps that you may find interesting; so feel free to try some of
them out. Note hat we have not installed Cytoscape, so we cannot visualize OTU
networks.
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We will end this tutorial with a 15-minute summary of what we have done and how
well our analysis compares with the one in the paper.
Hopefully you will have acquired new skills that allow you to tackle your own
taxonomic analyses. There are many more tutorials on the QIIME website that can
help you pick the best strategy for your project (http://qiime.org/tutorials/). We picked
QIIME for this tutorial as it is widely used and supported, but there are alternatives
that might suit your need better (e.g. VAMPS at http://vamps.mbl.edu; mothur at
http://www.mothur.org and others).

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